Methods for manufacturing sensors for sensing airborne contaminant of tobacco smoke

ABSTRACT

A method for manufacturing a sensor for sensing an airborne contaminant of tobacco smoke includes forming interdigitated electrodes on a substrate and depositing, on the substrate above the two interdigitated electrodes a conductive polyaniline bulk film having resistance sensitive to binding of the airborne contaminant of tobacco smoke thereto, such that resistance of the conductive polyaniline bulk film is measurable using the interdigitated electrodes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 13/495,258, filed Jun. 13, 2012, which is acontinuation-in-part application of PCT/US2011/051169, filed Sep. 12,2011, which claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 61/466,101 filed Mar. 22, 2011 and from U.S.Provisional Application Ser. No. 61/381,512 filed Sep. 10, 2010. Each ofthe aforementioned references is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Molecular imprinting is a technique that allows for the production ofmolecule specific receptors that are analogous to biological receptorbinding sites without the cost or environmental sensitivity of thenatural systems (Shea (1994) Trends Polym. Sci. 2:166; Wulff (1995)Angew. Chem. Int. Ed. 34:1812; Mosbach & Ramstrom (1996) Biotechnology14:163; BelBruno (2009) Micro and Nanosystems 1:163). Molecularlyimprinted polymers (MIPs) may be based on either covalent ornon-covalent binding between the host polymer and the target or templatemolecule. Various MIP-based devices have been suggested for use in thedetection of surface-binding molecules, inorganic compounds, organiccompounds, polymers, biological molecules, nanoparticles, viruses, andbiological arrays (WO 2008/063204 and US 2009/0115605).

Nicotine is a characteristic component of tobacco smoke and cotinine isa major metabolite of nicotine that is detected in the urine of smokers.Other reports of nicotine MIPs have appeared in the literature. Forexample, nicotine-targeted MIPs based on the synthesis of the polymerfrom methacrylic acid monomers have been reported (Sambe, et al. (2006)J. Chromatog. A 1134:88; Thoelen et al. (2008) Biosensors andBioelectronics 23:913-918). However, poly(methylacrylic acid) exhibitssolvent incompatibility with nicotine, thereby making the production ofthin films challenging.

SUMMARY OF THE INVENTION

The present invention is a device for monitoring exposure to airbornecontaminants. In one embodiment, the device is composed of at least twopoly(4-vinylphenol) or nylon films, each molecular imprinted with anairborne contaminant; a sensor for detecting binding between theairborne contaminant and the film, and a radio frequency interrogatorunit to read the sensor and transmit an interrogation signal. In otherembodiments, the sensor is a capacitive or conductive sensor, e.g.composed of polyaniline or polycarbozole. In particular embodiments, thepoly(4-vinylphenol) or nylon film is produced by phase inversion-spincoating. In another embodiment, the device is composed of a polyanilineconductive sensor, molecular imprinted with an airborne contaminant, anda radio frequency interrogator unit. In certain embodiments, theairborne contaminant is selected from the group of CO, nicotine,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and formaldehyde. Methodsfor monitoring exposure to airborne contaminants using a device of theinvention is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of an RFID circuit of the instant device.

FIG. 2A-2C is a schematic drawing showing sensor placement over thecircuit.

FIG. 3A shows the response of the sensor to vapor phase nicotine fromliquid nicotine held at a series of different temperatures; diamond=22°C., square=55° C., triangle=80° C.

FIG. 3B shows a plot of signal as a function of nicotine vapor pressure.

FIG. 4 shows the response of the sensor in terms of relative resistanceto exposure of vapor phase nicotine generated from a single cigarette inthe Teague system. Note also, that the sensor detects nicotine adsorbedon the walls in the chamber from previous experiments, so called thirdhand smoke.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a MIP-based personal sensor that can be readilyinterrogated using radio frequency identification (RFID) technology foruse in simultaneously monitoring airborne contaminants, such as thosefrom second-hand tobacco smoke. The personal monitoring device issimilar to the small badges used to monitor radiation doses and can bemonitored locally such that immediate feedback on exposure is provided.The specific airborne molecules detectable with the instant deviceinclude, but are not limited to, CO, nicotine,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and formaldehyde.Quantification of one or a combination of these components ofsecond-hand smoke can indicate that second-hand smoke is present.

In some embodiments, the device is composed of at least twopoly(4-vinylphenol) (PVP) and/or nylon films, each molecular imprintedwith an airborne contaminant; a sensor; and a radio frequencyidentification component. As is conventional in the art, molecularimprinting is a process by which guest or host molecules (functionalmonomers or polymers) are allowed to self-assemble around a moleculartemplate, thereby forming a recognition element, which has binding sitescorresponding to functional groups in the template molecule. Therecognition elements form a binding cavity which is cross-linked into amatrix. The template molecule is removed, leaving behind amolecularly-imprinted polymer (MIP) complementary in shape andfunctionality to the template molecule, which will rebind chemicaltargets identical to the original molecular template. In this invention,the host molecule is PVP or nylon composite, which non-covalently bindsthe template molecules, has solvent compatibility with the templatemolecules and is capable of forming a binding cavity around airbornecontaminants. For use in this invention, the nylon can be any nylonconventionally used in preparing molecular imprinted films and includes,but is not limited to, nylon 6 and nylon 6/6.

Thin films of the invention can be produced by any conventional method.However, the ability to control the thickness and formulate the films inan environment typical of printed circuit production is an importantfeature of film production for the instant sensors. Thus, in particularembodiments, the instant films are produced by phase inversion-spincoating onto a suitable substrate. The wet phase inversion procedure(Wang, et al. (1997) Langmuir 13:5396; Shibata, et al. (1999) J. Appl.Poly. Sci. 75:1546; Trotta, et al. (2002) J. Membr. Sci. 201:77) forpreparation of MIPs involves a polymerized starting material that isdissolved with the template in a theta solvent. A template-host networkis allowed to form in solution and precipitated by immersion in anon-solvent. Originally developed to produce MIP membranes, thisprocedure has been adapted to the production of thin, 300 nm to 5 μm,films via spin coating (Crabb, et al. (2002) J. Appl. Polym. Sci.86:3611; Richter, et al. (2006) J. Appl. Polym. Sci. 101:2919; Campbell,et al. (2009) Surface and Interface Analysis 41:347) and hydrogen bondinteractions between the template and host polymer.

By way of illustration, thin films containing PVP can be produced bymixing PVP (e.g., 10%-15% by weight) in conventional casting solutionwith the template molecule (e.g., about 5%-10% by weight) in a suitablesolvent. For example, nicotine is readily dissolved in methanol, whereasdimethylformamide (DMF) is a suitable solvent for formaldehyde and NNK.The solution is allowed to mix at room temperature, e.g., from six to 24hours, to form the hydrogen-bonded network in solution. Subsequently,thin films are cast onto a substrate using a spin coater at 5000-7000rpms for about 30 seconds. The thin film is allowed to dry and thetemplate molecule is removed by washing with water. In accordance withthis invention, a separate molecular imprinted film is produced for eachtemplate molecule so that detection in the assembled device occursindependently for each airborne contaminant. In this respect, certainembodiments of the device feature at least two films each independentlymolecular imprinted with CO, nicotine,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or formaldehyde.

The substrate of the molecular imprinted film can be a rigid or flexiblematerial, which may be conducting, semiconducting or dielectric. Thesubstrate can be a monolithic structure, or a multilayer or othercomposite structure having constituents of different properties andcompositions. Suitable substrate materials include quartz, glass,alumina, mica, silicon, III-V semiconductor compounds, and othersuitable materials. Optionally, additional electronic elements may beintegrated into the substrate for various purposes, such as thermistors,integrated circuit elements or other elements.

To detect an interaction (i.e., binding) between a molecular imprintedfilm and a template molecule (i.e., airborne contaminant), the devicefurther includes one or more sensors. Any suitable electrical propertymay provide the basis for sensor sensitivity, for example, electricalresistance, electrical conductance, current, voltage, capacitance,transistor on current, transistor off current, and/or transistorthreshold voltage. In the alternative, or in addition, sensitivity maybe based on a measurements including a combination of properties,relationships between different properties, or the variation of one ormore properties over time. In some embodiments of this invention, thesensor is a capacitive sensor, a conductive sensor or a combinationthereof. Depending on the type of sensor, the sensor can be a separateelement of the device or integrated with the molecular imprinted film.

Capacitive sensors are well-known in the art and any suitable sensor canbe employed. For example, the capacitive sensor can have a sandwich-typeelectrode configuration, wherein the molecular imprinted film is placedbetween two capacitor elements or electrodes. The electrode material canbe chosen from any suitable conductor or semiconductor e.g., gold,platinum, silver, and the like. By way of illustration, the instantdevice can use a set of interdigitated electrodes with the molecularimprinted film coated onto the electrode assembly. Specifically, asandwich-type capacitive sensor can be produced by depositing chromiumon a glass, silicon or mica substrate by thermal evaporation. Thechromium is patterned by photolithography and treated, subsequently, bywet etching. An insulating SiO₂ layer with a thickness between 40 nm and200 nm is deposited onto the bottom electrode surface using anelectron-gun thermal deposition technique. Subsequently, the molecularimprinted polymer layer is spun coated on the substrate surface. In thefinal step, a Cr film with a thickness of 70 nm is deposited on themolecular imprinted polymer film surface by thermal evaporation,followed with patterning by photolithography and wet etching.

As indicated, this device can alternatively incorporate one or moreconductive sensors. In this embodiment, a conductive polymer can be usedsuch that the template molecule becomes the doping agent. Accordingly,in the presence and absence of the template molecule, the conductivityof the polymer will be different. Conductive polymers of use in thisembodiment of the invention are so-called it electron-conjugatedconductive polymers. For example, polyaniline or a derivative thereof,polypyrrole or a derivative thereof, polythiophene or a derivativethereof, or a copolymer of two or more kinds of these materials aresuitable conductive polymers. By way of illustration, polyaniline filmswere prepared for the detection of formaldehyde (see Example 3) andnicotine (see Example 4). Accordingly, in particular embodiments,airborne contaminants are detected using a conductive polymer such aspolyaniline or polycarbozole.

To remotely communicate the output of the sensors, the device furtherincludes a radio frequency interrogator unit. Such interrogator unitstypically include an antenna, and transmit an interrogation signal orcommand via the antenna. The instant device can operate over a widerange of carrier frequencies. For example, the device can operate withcarriers of 915-5800 MHZ, wherein the frequency selectivity is based onselection of the antenna. Moreover, in so far as the device employs oneor more MIP films to simultaneously detect multiple airbornecontaminants, the device further includes multiplexing RFID circuitry.The sensors of the instant device have a range of values and theinterrogator unit must be capable of reading two or more separatesensors (FIG. 1). As such, in certain embodiments, the instant devicealso has a multiplexing amplifier circuit that includes on-board memoryto store values. This includes assembling on-board memory and addressinghardware and certifying the operational status before calibration of thefinal device to assure that the assembled sensor functions within thesame parameters as the individual components. An example of an RFIDcircuit of the instant device is shown in FIG. 2A-2C.

Interrogator units also generally include dedicated transmitting andoptionally receiving circuitry. Active transmitters are known in theart. See, for example, U.S. Pat. No. 5,568,512, which also discloses howthe transmit frequency for the transmitter is recovered from a messagereceived via radio frequency from the interrogator. Moreover, manyexamples of wireless communications circuits are known in the art, andany suitable low-power circuit may be employed. The invention isintended to be practiced with any radio communications circuit with lowpower requirements, for example, a circuit appropriate for extendedoperation in a remote battery-powered device without need forrecharging.

The device of the invention can transmit radio frequency signals to areceiver and/or host computer in communication with the interrogator. Anexemplary receiver includes a conventional Schottky diode detector. Whena host computer is employed, the host computer can act as a master in amaster-slave relationship with the interrogator. The host computer caninclude an applications program for controlling the interrogator andinterpreting responses, and a library of radio frequency identificationdevice applications or functions. Most of the functions communicate withthe interrogator.

Although sensor systems described herein are particularly suitable forefficient operation by conventional power sources used inportable/remote electronics (e.g., battery, solar cell, miniature fuelcell), the instant device can also use alternative energy resources,such as a thermocouple, radio-frequency energy, electrochemicalinteractions, supercapacitors, energy scavenging mechanisms, or thelike, or combinations thereof.

In so far as the device can detect airborne contaminants such as CO,nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and/orformaldehyde, the device of this invention is of particular use inmonitoring exposure to second-hand tobacco smoke. Upon exposure of thedevice to an air sample, the molecular imprinted films bind airbornecontaminants in the air sample, sensors of the device sense saidbinding, and radio frequency signals are transmitted to alert the userof exposure to the airborne contaminants.

While conventional technologies can measure nicotine and particulatematter in indoor environments, the current technology involves large airsampling devices and hinders the process of immediate feedback, sincecomplex procedures are required, in an analytical laboratory, toquantify the samples. Moreover, the sampling technique collects allairborne material and the analytical instruments are required to sortout the adsorbed material. The device is unique in that it is small(personnel badge sized), easy to read, simple (no laboratory analysis isneeded), the MIP films are targeted to specific contaminants and anumber of contaminants can be simultaneously detected. As awall-mountable or wearable, personal badge-type, second-hand smokedetector, the instant device finds application in, e.g., medical centersto monitor the environment of pediatric (and other) patients, and tomeasure tobacco smoke contamination in supposedly smoke-freeenvironments such as hotel rooms and rental cars. Since the specificsensor in the monitor may be readily changed to monitor a differentair-borne contaminant, the instant device can be adapted to detect anarray of different molecules or hazardous vapors.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Production of PVP MIP Films

The aromatic nature and hydrogen bonding potential makePoly(4-vinylphenol) (PVP) an ideal host matrix for MIPs. The PVP filmswere produced by spin coating; a simple deposition technique that issensitive to the composition and viscosity of the solution and therotating speed of the plate (Bronside, et al. (1987) J. Imaging Technol.13:122).

Solutions composed of 10 mL of methanol (Acros Organics; ACS ReagentGrade 99.8%) with 10 wt % of PVP powder obtained from Polysciences, Inc.(MW=22,000; T_(g) 150° C.) and 5 wt % of nicotine or cotinine werenitrogen purged, covered, and stirred at room temperature for 24 hours.Control films (NIPs) were similarly produced, but without the nicotineor cotinine Films were spin cast from these solutions onto 22 mm squareglass microscope cover slips. Typically, the slides were prewashed withspectroscopic grade isopropanol and acetone prior to polymer deposition.The coating solution was dropped onto a stationary substrate and thespin coater was operated at 4000 rpm for 30 seconds with negligible rampup time. The rotation spreads the solution evenly over the surface andalso causes the solvent to evaporate leaving a thin film of material onthe substrate. The concentration of PVP in the casting solution is thedominant variable for the film thickness, which increases rapidly withincreasing concentration (solution viscosity). Cast films are quitestable and may be stored or used for an indefinite time.

The template molecule was removed from the film by immersion indeionized water for five hours. Nicotine (or cotinine) removal wasconfirmed by FTIR measurements. Template reinsertion (or reinsertion ofthe complementary template molecule) was accomplished by immersion ofthe template extracted (or control) film in a 5 wt % solution of themolecule in deionized water for 2.5 hours. This reinsertion, as with thetemplate removal procedure, is an equilibrium-controlled process andreinsertion occurs to approximately 50% of the initial concentration(via qualitative FTIR measurements). Additional immersion time was notfound to increase the relative amount of template molecule reinsertedinto the film. FTIR spectra were recorded over a narrow region ofinterest, ˜3400 cm⁻¹ for the OH stretch of PVP, which is missing whenhydrogen bonded to nicotine or cotinine or ˜1700 cm⁻¹ in the carbonylregion of cotinine, to confirm the interaction of the template with thepolymer in the film. The surface topography of the films ischaracterized by average roughness measurements, R_(a), using scanningforce microscopy (SFM). It is defined as the average deviation of theprofile from a mean line or the average distance from the profile to themean line over the length of the assessment. The surface roughness,R_(a), is given by the sum of the absolute values of all the areas aboveand below the mean line divided by the sampling length.

All nanoindentation experiments were performed using the electrostatictransducer of the Hysitron triboscope in the UBI 1 (Hysitron UserHandbook: Feedback Control Manual. 10025 Valley View Road, Minneapolis,Minn.; Hysitron, Inc.). The transducer is a three-plate capacitor, themid-plate of which carries the indenter fixed to a thin stylus.Application of a DC voltage generates an electrostatic force driving theindenter into the sample surface, while the capacitance change as ameasure of penetration depth is recorded. The data consist of aforce-displacement curve. For soft samples such as polymers, thestiffness of the internal springs holding the indenter must besubtracted from the applied load in order to obtain the samplestiffness. Hardness, H, is calculated as the applied load, F, divided bythe area, A_(c), of the indenter tip at the contact depth, v_(hc); thearea is depth-dependent (Olivier & Pharr (1992) J. Mater. Res. 7:1562).The modulus is derived from the slope of the force-displacement curveupon unloading when the sample elastically recovers. The tip elasticproperties can effectively be ignored for polymeric materials.Investigations are performed with a blunted 90° diamond cube corner tip.The calibration of the tip to determine the depth dependent areafunction A_(c)(hc) was obtained with the standard curve-fitting methodusing fused quartz with its known reduced modulus as the referencematerial. Additionally, calibration with a sharp silicon grating wasperformed (Richter, et al. (2006) High Pressure Res. 26:99). Acommercial grid with ultra sharp conical silicon tips was used. Thesmall apex angle of the grid tips (below 20°) together with their largeheight (700 nm) offers the opportunity for exact examination of theshape of the diamond indentation tip apex. By means of the AFM softwareusing the so-called bearing function, the shape of the diamond indentercan be reconstructed and the area function A_(c) can be obtained. Theadvantage of this technique over the curve-fitting method is the directobservation of the tip shape, which also allows the estimation of theblunt tip radius, which was 600 nm in our experiments.

Thermal drift and creep behavior of the piezoelectric scanner must beminimized. At the nanoscale, drift is measured and compensated in theresulting data. This compensation factor is part of the standard UBIsoftware and a correction measurement is performed before each indent.Typical drift rates range up to 0.5 nm/s. The penetration depth of theindent should not exceed 30% of the polymer film thickness to avoidsubstrate effects. Most experiments were performed with smallerpenetration depths, however depth-dependent measurements sometimes showan increase in hardness and reduced modulus with increasing penetrationindicating the influence of the glass substrate.

Depth-dependent mechanical properties are obtained through indentationtests where repeated loading and unloading are performed at the samelocation on the sample surface (Richter, et al. (2006) Colloids andSurfaces, A 284/285:401; Fischer-Cripps (2002) Nanoindentation,Springer, New York; Olivier & Pharr (1992) supra; Wolf & Richter (2003)New J. Phys. 5:15.1; Maier, et al. (2002) Mater. Character. 48:329; Ward& Hadley (1993) An Introduction to the Mechanical Properties of SolidPolymers, John Wiley & Sons, Chichester; Nowicki, et al. (2003) Polymer44:6599; Du, et al. (2000) Polymer 42:5901; Drechsler, et al. (1998)Appl. Phys. A 66:825; Tsui, et al. (2000) Macromolecules 33:4198;VanLandingham, et al. (2001) in: Tsukruk & Spencer (Eds.) MacromolecularSymposia, Wiley-VCH Verlag, Weinheim, pp. 15-43). Eight cycles ofmulti-indentation were performed to calculate the depth-dependenthardness and the indentation modulus. In general, multi-cycling means,after loading to a maximum load, F_(n), the sample is partially unloadedto a minimum load, F_(min)=0.1 F_(max) to 0.25 F_(max), required toprevent the tip from losing contact with the sample and sliding to a newlateral position. The sample is then reloaded to the same or anincreased maximum load (F_(max)+ΔF) and the cycle is repeated. After theonset of plastic deformation, the loading curve is an overlap of bothplastic and elastic deformations. Multi-cycling delivers a set of datathat includes the entire material response, from the firstindenter-sample contact to the maximum penetration.

Computational Details.

All optimizations were performed with NWChem, a Computational ChemistryPackage for Parallel Computers, v5.1, with no symmetry or geometricconstraints. The correlation and exchange effects were calculated usingthe Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional (Adamo& Barone (1998) J. Chem. Phys. 110:6158) with the 6-31G* basis set(Hariharan & Pople (1973) Theoret. Chimica Acta 28:213) both for allatoms. Several relative orientations of the PVP molecule(s) relative tonicotine or cotinine were optimized to ensure that the total energy ofthe complex was not dependent upon this factor. All calculations wererun in parallel on a Linux cluster composed of 94 Quad-Core (2×) AMDOpteron nodes (752 cpus), and 6 Quad-Core (2×) Intel nodes (48 cpus). Inaggregate, the Linux cluster had 3 terabytes of memory and more than 35terabytes of disk space. Geometric structures were visualized using theAVOGADRO molecular editor program.

Example 2 Nanohardness Analysis of Nicotine- and Cotinine-ImprintedPoly(4-vinylphenol) Films

Control PVP and MIP Film General Features.

The structure of the MIP and NIP films was dependent on the viscosity ofthe solution. This was mainly controlled by the temperature and spincasting conditions such as speed and deposition time, in addition to thePVP concentration in the solution. The pure PVP films deposited from thecasting solution containing 10% polymer had a characteristically smoothmorphology. In the present analysis, the pure PVP film had a surfaceroughness, R_(a), of 11.5 nm, over a 130 μm×130 μm sample. Nosignificant morphological features were found in the control PVP films;the films were flat. The ‘as produced’ MIP films containing, for examplenicotine template molecules, showed a different surface morphology incomparison to the control films. Surface stripes, representing differentheights, were the main surface feature. The surface roughness of thistype of sample was measured to be 69.2 nm, over the same 130 μm×130 μmsampling size. Removal of the nicotine from the MIP resulted in a lossof the stripe morphology and the observation of a number of pores in thesurface. The pores were apparently formed during the solidificationprocess of the polymer films and were caused by the presence of thetemplate molecules and the porogen solvent during the film growthprocess (Campbell, et al. (2009) supra). The assumption was that thepores were present in the ‘as produced’ samples, but lay beneath thestripe morphology. The template molecules were smaller than the size ofthe pores observed in the films. The additional volume of the measuredpores resulted in part from: the geometrical form of the templatemolecule, the arrangement of that molecule within the polymer host, andthe evaporation of the solvent through the polymer film. The surfaceroughness R_(a), of the nicotine-removed MIP was 44.1 nm, over the 130μm×130 μm sample. Reinsertion of nicotine into this MIP restored thestrip morphology somewhat, but had minimal effect on the roughness ofthe surface (R_(a)=33.1 for the 130 μm square sample). The differentfilm morphology in the SFM images was characteristic for the presence ofthe template molecules.

Nanomechanical Properties.

The contact pressure (hardness) can vary even for homogeneous mattersuch as the control sample, since the deformation starts with purelyelastic deformation, and after yielding, the plastic contributionsincrease after a saturation value of H is obtained for very largeindents. This means the hardness decreases with increasing indentationdepth. Within the indentation size effect model (Wolf & Richter (2003)supra) the hardness will be higher in a small indentation area wherefewer defects are encountered. With increasing indentation size (depth),more defects such as dislocations, are generated by the contactpressure. For very thin polymer films, the indentation modulus is notconstant due to the increased substrate influence with increasing depth.The elastic behavior of pure PVP films occurs by deformation of thepolymer molecules and movement of the chains after the adhesion energyhas been overcome.

The multi-cycling load-depth curves for MIP films with templatemolecules in the casting solution for the spin coating process showedsignificant differences in comparison to pure PVP films. From theload-depth curves, it was clear that the ‘as produced’ nicotine-loadedMIP films had indentation depths of approximately 175 nm with a maximumapplied force of 300 μN; removal of the nictoine increased thepenetration depth to nearly 200 nm Unexpectedly, reinsertion of nicotinereduced the penetration depth to a value 35 nm less than the originalimprinted film. Clearly, the presence of the template molecule in theMIP led to a stiffer film and analogous results were recorded forcotinine imprinted films. The assignment of the basis of the change innanomechanical properties to the template was reinforced by the factthat the MIP with the template removed was less stiff than a pure PVPfilm for the same applied force.

The nanomechanical behavior of the polymer films indicated that hardnessdecreased slightly with increasing depth, whereas the indentationmodulus increased slightly with increasing depth. The hardness of thecontrol PVP film had the value of 0.38 GPa with an indentation modulusof 11.7 GPa. MIP films with cotinine were stiffer with a hardness valueof 0.59 GPa and an indentation modulus of 14.7 GPa. Nicotine imprintedfilms were slightly stiffer than the control film with a hardness valueof 0.43 GPa and a modulus of 11.6 GPa. Extraction of the templatemolecules meant that the molecular cavities were still in the polymermatrix, but the space was empty. Thus, the network character andtherefore the mechanical properties changed. For example, the hardnessfor MIP films with nicotine extracted yielded smaller values of 0.31 GPafor the hardness. Reloading of either template resulted in an increaseof the hardness to values greater than those of the original ‘asproduced’ MIP films. The percentage increase in hardness was greater forthe reinsertion of nicotine into a nicotine-targeted MIP than forcotinine into a cotinine-targeted MIP. Loading, extraction and reloadingof nicotine or cotinine in the MIP films were clearly measurable withthe nanoindentation method.

These results indicate a strongly hydrogen-bonded network between thepolymer chains via the template molecules. In the ideal case, themolecular cavities with nicotine template molecules were formed by twohydrogen bonds that cross-linked between two PVP molecules; cotinine hadthree such potential hydrogen bonding sites. However, the imprintingprocess could be incomplete with fewer hydrogen bonds established.Template molecules can bond to the polymer molecule at several pointsalong the chain with efficiencies dependent on the number anddistribution of template molecules in the MIP film. From thenanomechanical investigations it was contemplated that hydrogen bonds ofthe template molecules between the PVP chains resulted in across-linking between the chains, separated the PVP molecules,(preventing an easy movement of the chains, and could reduce theadhesion energy between pure PVP molecules. This could result in either,mechanically stiffer or softer MIP networks. Two different molecularmechanisms were proposed for the polymer response during an appliedexternal contact pressure. In pure PVP, the indentation tip could causea deformation of the PVP molecules and a sliding motion between thechains. The molecular cavities and micro-pores change the mechanicalproperties in two directions compared to pure polymer films. Filledcavities (template-loaded MIP) showed an increase in hardness incomparison to pure PVP films. This meant a stiffer molecular network wasestablished. In MIP films, the chains are fixed by hydrogen bonds andthe sliding motion is inhibited in general. The filled molecularcavities prevent strong elastic deformation. Empty cavities afterextraction of the template, result in a large decrease in the hardness.This could be caused by the fact that the empty MIP network can beeasily squeezed together, resulting in a lower hardness in comparison tothe pure polymer network. In MIP films, the PVP chains are fixed by thehydrogen bonds and the formed molecular cavities, but the deformationaround the empty cavities is flexible (breathing cavities). Therefore,no gliding motion of the chains occurs. This means, the main effect forthe change of the mechanical properties in different stages of MIP filmsoriginates from the formed molecular cavities. If they are filled withthe template molecule the material is harder; if the cavities are empty,the compression of the cavities leads to a much softer material. Theelastic compression of the empty cavities and pores acts in the samedirection as the mechanism of deformation and gliding of the PVP chains.

Computational Study of Hydrogen Bonding.

Reports in the literature indicate the usefulness of computationalchemistry in selecting a polymer host for MIP development with aparticular target molecule (Breton, et al. (2007) Biosens. Bioelectron.22:1948). However, it was contemplated that computational studies wouldbe of use in providing information to understand experimental MIPresults. The optimized structures of nicotine and cotinine were firstobtained for reference. These molecules differ only by the addition of acarboxyl group on the pyrrolidine ring of cotinine. The geometry of theplanar pyridine ring, including bond lengths, in the two molecules isidentical. The presence of the oxygen atom in cotinine results in bondshortening in the pyrrolidine ring, as well as a further distortion fromplanarity relative to the nicotine pyrrolidine ring. The pyrrolidinenitrogen becomes more sp2-like rather than the pyramidal angle observedin the nicotine molecule. As a final reference point, the 4-vinylphenoldimer structure was optimized and was found to have a hydrogen bondlength of 1.876 Å and a hydrogen bond energy of 0.34 eV. The geometricparameters of the 4-vinylphenol molecule were unchanged in the dimer.

Nicotine has two potential hydrogen binding sites and cotinine has threesuch sites. In both clusters, the 4-vinylphenol geometry was identicalto that of the unbonded molecule and the pyridine rings bond lengthswere unaffected by the hydrogen bond. The hydrogen bond from 4VP to thepyridine nitrogen in nicotine had a length of 1.822 Å, while that to thepyrrolidine nitrogen was 1.788 Å. The C—N bond lengths in this ring bothincreased. The total hydrogen bonding energy was 0.84 eV. In cotinine,only two hydrogen bonds formed. The bond to the pyridine nitrogen had alength of 1.820 Å and that to the carboxyl-oxygen was 1.784 Å with atotal hydrogen bonding energy of 1.00 eV. Attempts to add a thirdhydrogen bond at the pyrrolidine nitrogen site failed, as the additionalPVP molecule was repulsed from the ring. The C—N bond lengths in thepyrrolidine ring both decreased upon hydrogen bonding. Finally, it wasnoted that attempts to obtain a π-π complex between cotinine and 4VPindicated that the ring interactions were repulsive. Clearly, the DFTcalculations indicated that cotinine would complex to the PVP hostmatrix with a greater binding energy than would the nicotine templateand the experimental results reflected the results of thosecalculations.

Example 3 Preparation of Polyaniline-Nylon Films

Polyaniline has the general structure:

Polyaniline was selected for the film given its conductivity (Scheme 1).

Using phase inversion, formaldehyde cavities were created in apolyaniline (PANI)-Nylon 6 composite film. Films were produced bydissolving 0.2 g of PANI, 0.2 g of Nylon 6 and 200 μl of formaldehyde informic acid. The formic acid dissolved the composite and formaldehyde toform a rigid polymer-formaldehyde network. After sufficient mixing, theimprinted polymer solution was uniformly spin-coated onto a glasssubstrate to form a thin film. After making the formaldehyde imprintedfilms, the formaldehyde molecules were extracted from the film aerially,leaving behind formaldehyde-specific receptor sites that were capable ofmolecular recognition and binding of formaldehyde molecules withremarkable specificity.

Infrared spectra analysis conclusively indicated that formaldehydemolecules could bind to the PANI-Nylon 6 composite through stronghydrogen bonding due to the presence of an elongated carbonyl group at1722 cm⁻¹. This peak was present in the imprinted polymer composite andnoticeably absent in the control. This analysis indicated thatPANI-Nylon 6 was successfully imprinted with formaldehyde. Moreover, theintensity of the peak indicated the efficacy of the imprinting process.

Changes in electrical resistance of imprinted polymer and controlpolymer following controlled exposure to formaldehyde vapor weredetermined using lithographically patterned interdigitated electrodes.The results of this analysis indicated that because the imprintedpolymer had formaldehyde-specific cavities, it was able to selectivelyadsorb the formaldehyde molecules, which caused a dramatic increase inresistance of the film. In contrast, the control film with no cavitiesexhibited a relatively insignificant increase in electrical resistancein response to the formaldehyde vapor.

Example 4 Preparation of Polyaniline Films in Sensors for DetectingNicotine

Materials and Methods.

Polyaniline was purchased from Polysciences, Inc. as the undopedemeraldine base form with a molecular weight of 15,000 and aconductivity of 10⁻¹⁰ S/cm. Formic acid, >98%, was purchased from EMDChemicals and used to dissolve the polyaniline prior to spin casting.Secondary doping increased the sensitivity of the films and HCl,purchased from Fisher Scientific (ACS Certified), was used in a 1.0 Maqueous solution. For laboratory studies, nicotine purchased fromAlfa-Aesor, 99%, was used. All reagents were used as received withoutany further treatment. The standard cigarettes used in the smokingchamber were 3RF4 reference cigarettes, containing ˜0.8 mg of nicotine.

The polymer films for detecting nicotine were spin-cast polyaniline.Polyaniline in its conductive form is insoluble. However, the emeraldinebase may be dissolved in several solvents, including the 98% formic acidused herein. The spin casting solution was produced from formic acid asa 1% (by weight) polymer solution. Because the pK_(a) of formic acid is3.77, polyaniline in this solution was 50% protonated; the amine andimine nitrogen atoms had different pK_(a) values. To complete theprotonation process and increase the sensitivity of the film, secondaryprotonation in 1.0 M HCl was employed. Protonated solutions are green,while solutions of the base are deep blue. Morphology and roughness wereinvestigated by atomic force microscopy using a Pacific NanotechnologyNano-1 microscope in close contact mode.

The conductive sensors were constructed on oxidized silicon substratesusing chromium metal with a nickel overlayer for the electrode and theprotonated polyaniline film as the active element above the electrode.The electrode was patterned into an interdigitated grid with 40 μmfingers and 20 μm spacing.

Prime grade silicon wafers with a 5000 Å thermally deposited oxide layerwere used for the substrate. These films were patterned byphotolithography and subsequently wet-etched to produce the finalelectrodes with a total area of 376 mm², following vapor deposition of200 Å of chromium and a 1000 Å overlayer of nickel. Liftoff wasaccomplished using acetone, with final rinses of water.

Subsequently, the polyaniline polymer layer was spun on the sample. Analiquot of 0.5 ml of solution was dropped onto the substrate (oxidizedsilicon), and allowed to spread for 20 seconds. The spin-coater was thenbrought up to 4000 rpm for 30 seconds. This resulted in deposition offilms with a typical thickness of approximately 100 nm. In the finalstep, secondary doping with 1.0 M HCl was accomplished by dip-coatingfor 30 seconds. After this treatment, background (washed) resistancevalues were measured, and the sensor was ready for use in bindingstudies.

Smoking machine experiments were carried out in a Teague Enterprisespackage (Teague Enterprises, Davis, Calif.), composed of a TE-10 smokingsystem and a mouse exposure system. The smoking device wasmicroprocessor controlled and produced both mainstream and sidestream(separately or simultaneously) smoke from filtered research cigarettesproduced with controlled nicotine content. Up to ten cigarettes could besmoked simultaneously following the Federal Trade Commission procedureand expended cigarettes could be automatically extinguished and ejected.Smoke was captured and transferred to a mixing chamber for exposureexperiments; sidestream or mainstream smoke was mixed with air and thenpassed into the exposure chamber. However, for the experiments describedhere, the system including sample lighting and extinguishing wasoperated in manual mode. A filter was available for venting and purgingthe system. The exposure chamber was calibrated for total suspendedparticles (TSP), carbon monoxide and nicotine concentration determinedfor selected mixing valve and fan settings. All measurements using theTeague Enterprises system were made with the polymer sensors in theexposure box, using calibrated operational parameters.

The laboratory sample system was composed of a small nylon box,containing spring-mounted electrodes and a small (˜3 cm³) well filledvia a syringe through a septum. The sensor assembly was placed on theelectrodes above the well and a nylon cap was attached using a torquewrench to ensure reproducible pressure of the sensor against thespring-mounted electrodes. Nicotine (1 mL) was injected into the welland the response of the sensor was recorded. To follow the recovery ofthe sensor after exposure to nicotine, dry nitrogen was passed throughthe well to evaporate the nicotine. In both experimental chambers, thechange in the resistance of the sensor was measured using a multimeterconnected to a laboratory computer.

The resistance, R, of the polymer sensor was measured using a KeithleyModel 2100 6½ Digit Multimeter. During the measurement, constant currentof 1 mA was applied and the voltage through the film was recorded,providing a resistance value via Ohm's law. Total dissipated powerwithin the sensor was less than 2 W. Four point measurements were foundunnecessary and all of the reported data were obtained using twocontacts. Data were taken at a rate of 1 Hz over as long as 9 hours, buttypically over considerably shorter times. The resistance increased fromits low background value prior to exposure, typically 600 □, through toa plateau, associated with the level of nicotine in the sample chamber.Data are reported as normalized resistance, referenced to the initial,out of chamber background value.

Films were exposed to analyte concentrations that ensure a challenge tothe adsorption process. The results provided an indication that theshift in the resistance value and the rate of change in the resistance,were proportional to the quantity and identity of the analyte adsorbed.

Results and Discussion.

The morphology of the film surface was investigated by atomic forcemicroscopy (AFM) of films produced on both silicon oxide and glass underthe coating conditions described above. The undoped film was rougherthan the doped material and more irregular with surface defects. Thedoped film was somewhat smoother and the minimal occurrence of surfacedefects provided an ideal material for adsorption of the target moleculefrom the vapor phase.

The physical property associated with the target molecule presence inthe film was the increase in the resistance. Sensor functionalitydepended upon detecting differences in this property as a function ofthe adsorption of the target nicotine onto the sensor chip. Numerousfilms were tested using both pure nicotine in the small lab-builtchamber and nicotine emitted from cigarette consumption as measured inthe Teague smoking system. Data presented here are typical of theseobservations.

Testing of the sensor in the laboratory chamber indicated that injectionof nicotine into the sample well evoked an immediate rise in themeasured resistance. Nicotine vapor pressure was quite small at roomtemperature, so a series of experiments, injecting nicotine at differentinitial temperatures (providing different vapor pressures and, hence,vapor phase concentrations of nicotine in air) and recording theresistance was completed. The relationship of nicotine vapor pressure tosample temperature is well established and was used in this analysis(Young & Nelson (1928) Ind. Eng. Chem. 20:1381-1382). The results ofthis study are shown in FIG. 3A for three different noiminaltemperatures. For example, consider the film response to the injectionof nicotine at a nominal 80° C. The rise of the signal as the sample wasinjected and the beginning of a plateau of the signal (and slightdecrease) as the sample cooled was clearly demonstrated. FIG. 3B shows aplot of the signal (15 seconds post injection) as a function of thenicotine vapor pressure at the nominal temperatures. A linear fit to thedata with a correlation coefficient of 0.99 is shown. The nicotine beganto cool almost immediately, therefore, deviation of the fit from anexact correlation with temperature was to be expected. The absence ofconstant temperature capability in this device precluded its use as acalibration system. However, the trend of increasing resistance withincreasing temperature was clear and demonstrated the responsiveness ofthe film to pure nicotine. The nicotine concentrations in this devicewere estimated to be of the order of a few ppm.

FIG. 4 shows the time evolution of the sensor film signal for smoking asingle cigarette in the Teague system. The system calibration at theinflow/outflow settings of the exposure chamber provides that thedynamic nicotine concentration in this situation from the cigaretteconsumption alone was 0.5 ppb. The actual concentration was, of course,higher since there was clearly background nicotine adsorbed onto thechamber surface. In general, the resistance increased as the cigarettewas consumed, increasing by 50% over the signal assigned to the chamberbackground. It was interesting to note that the background reading ofthe sensor, the resistance at the zero time point, immediately increasedby 20% as the film was placed into the exposure chamber, indicating abackground level of nicotine before engaging the smoking apparatus.Prior to this experiment, the smoking chamber had been in constant usefor 8 hours and deliberately not cleaned in the 2 hours prior to itsapplication in the current experiment. The sensor was capable ofmeasuring nicotine that was outgassing from the plastic chamber walls,an event labeled as “third hand smoke” when this event occurs ininhabited rooms and automobiles (Sleiman, et al. (2010) PNAS107:6576-6581). During the smoking process, sidestream smoke was fedinto the exposure chamber and, as long as the smoking was continued, theresistance increase indicated adsorption of nicotine in to the film. Thesignal stopped increasing as the cigarette was extinguished anddecreased as air blown into the exposure chamber from the smoking systemcontained no additional nicotine component. After approximately sixminutes, the chamber was purged with 100% fresh (room) air and thesensor resistance dropped accordingly to a level approximately 20% abovethe chamber background.

A set of sequential exposures in the Teague system, using varied numberof simultaneously smoked cigarettes followed by a brief fresh airblowout was conducted. Cigarettes were smoked over a period of eightminutes during which the sidestream smoke filled air from the smokingdevice was mixed with an equal volume of fresh air and fed into theexposure chamber. Following the extinguishing of the consumed cigarette,fresh air was blown into the exposure chamber for a period of sixminutes. The fresh air phase was shown to allow at least some of thenicotine to be removed from the sensor and restore the resistancemeasurement to a smaller value. These two different regimes were clearlydiscernible. The fresh air phase of the repetitive experiment was notsufficient to bring the sensor back to the original baseline. That is,the process of removing the nicotine from the film was slower than thetime used in the study and subsequent exposures included increasedresidual levels of nicotine from the walls of the chamber in addition tonicotine that remained on the sensor film from the previous cigaretteconsumption. However, the final exposure cycle, with a longer smoke-freeperiod, indicated that a return approximately to the original baselinewas possible. Indeed, a resistance measurement made several hours aftercompleting the experiments resulted in a value nearly equal to theinitial resistance. The slopes of the rising signals were also relatedto the number of cigarettes simultaneously consumed and, hence, theconcentration of nicotine in the chamber. The system provided a dynamicconcentration of 0.75 ppb and 1.11 ppb, for sidestream smoke generatedsolely by two and three cigarettes, respectively. But as describedearlier, this underestimated the true nicotine concentration. It wasnoted that the first, single cigarette consumed increased the signal by˜60% and second, consecutive single cigarette furthered the increase by32%. The next sample involved two cigarettes and resulted in a 40%signal increase with a final sample of three cigarettes and a 42%increase in resistance. The “blow out” phase returned the signal toapproximately the resistance measured after the first experiment.

To test the response and recovery in a heavy smoking situation, severalsuccessive runs were conducted in which 10 cigarettes (nominallyproviding 3.16 ppb of nicotine) were simultaneously smoked. The sensorbackground resistance was measured in ambient room air prior toinsertion in the exposure chamber, providing a clear visualization ofthe ability to detect nicotine from the chamber walls. The firstcigarette burn resulted in a steep increase in resistance. After a sixminutes delay (following cigarette extinguishment), a new burn wasbegun, followed by two additional ten cigarette exposures. The absenceof significant recovery time post-exposure decreased the absoluteincrease in signal (although the third and fourth exposures providedsimilar increases) with measured changes in resistance of 110%, 25%, 15%and 9%. The fresh air purge at the end of the experiment did lower thesignal substantially. Most importantly, this particular study indicatedthat the film is sensitive to its environment, even if the ambientatmosphere has a relatively heavy concentration of smoking generatednicotine.

This analysis demonstrated that a simple chemiresistor based on apolyaniline film and interdigitated electrode can monitor nicotine toprovide a real time indication of exposure to second hand cigarettesmoke. The polyaniline film was shown to be sensitive to the number ofcigarettes consumed, demonstrated reasonable recovery between exposuresand was functional in the presence of simulated heavy smoking. Thedetection of nicotine outgassing or “third hand smoke” was alsodemonstrated to be feasible using the polymer film assembly.

What is claimed is:
 1. A method for manufacturing a sensor for sensingan airborne contaminant of tobacco smoke, comprising: forminginterdigitated electrodes on a substrate; dissolving bulk polyanilineand the airborne contaminant of tobacco smoke in a protonating agent toform a partially protonated and conductive polyaniline solution;depositing the partially protonated and conductive polyaniline solutionon the substrate above the interdigitated electrodes to form aconductive polyaniline bulk film that is partially protonated; anddoping the conductive polyaniline bulk film with a secondary dopant tofully protonate the conductive polyaniline bulk film, so as to form thesensor.
 2. The method of claim 1, the step of dissolving comprisingdissolving the bulk polyaniline and the airborne contaminant in aprotonating agent that is at least 98% pure.
 3. The method of claim 2,the step of dissolving comprising dissolving the bulk polyaniline andthe airborne contaminant in formic acid.
 4. The method of claim 1, thestep of doping comprising doping the conductive polyaniline bulk filmwith HCl.
 5. The method of claim 1, the step of doping comprisingdip-coating the conductive polyaniline bulk film in the secondarydopant.
 6. The method of claim 1, the step of depositing comprisingspin-coating the partially protonated and conductive polyaniline bulksolution onto the substrate.
 7. The method of claim 6, the step ofspin-coating comprising forming the conductive polyaniline bulk filmwith a thickness of approximately 100 nanometers.
 8. The method of claim1, the step of forming interdigitated electrodes comprising forming twointerdigitated electrodes separated apart by approximately 20 microns.9. The method of claim 8, the step of depositing comprising forming theconductive polyaniline bulk film with a thickness of approximately 100nanometers.
 10. The method of claim 1, the airborne contaminant beingnicotine.
 11. A method for manufacturing a sensor for sensing anairborne contaminant of tobacco smoke, comprising: forming twointerdigitated electrodes on a substrate, the two interdigitatedelectrodes being separated apart by approximately 20 microns; on thesubstrate above the two interdigitated electrodes, depositing aconductive polyaniline bulk film having resistance sensitive to bindingof the airborne contaminant of tobacco smoke thereto, such thatresistance of the conductive polyaniline bulk film is measurable usingthe two interdigitated electrodes.
 12. The method of claim 11, the stepof depositing comprising producing the conductive polyaniline bulk filmwith thickness of approximately 100 nanometers.
 13. The method of claim12, the step of depositing comprising spin-coating a conductivepolyaniline solution onto the substrate above the two interdigitatedelectrodes.
 14. The method of claim 11, the step of depositingcomprising forming each of the two interdigitated electrodes with fingerwidth of approximately 40 microns.
 15. The method of claim 14, the stepof depositing comprising producing the conductive polyaniline bulk filmwith thickness of approximately 100 nanometers.
 16. The method of claim15, the step of forming comprising forming each of the twointerdigitated electrodes with thickness of approximately 120nanometers.
 17. The method of claim 11, the step of depositingcomprising depositing a fully protonated conductive polyaniline bulkfilm on the substrate.
 18. The method of claim 11, in the step ofdepositing, the conductive polyaniline bulk film being molecularlyimprinted with the airborne contaminant.
 19. The method of claim 11, theairborne contaminant being nicotine.
 20. The method of claim 11, furthercomprising measuring background resistance between the twointerdigitated electrodes when the device is not exposed to the airbornecontaminant.